Process for the preparation of recombinant polypeptides

ABSTRACT

The present invention relates to the preparation of a recombinant polypeptide, which polypeptide upon expression has been secreted into the periplasm of a transformed host cell. In particular, this invention relates to a process to enhance the extraction yield of said recombinant polypeptide from the periplasm before further downstream processing.

FIELD OF THE INVENTION

The present invention relates to the preparation of a recombinantpolypeptide, which polypeptide upon expression has been secreted intothe periplasm of a transformed host cell. In particular, this inventionrelates to a process to enhance the extraction yield of said recombinantpolypeptide from the periplasm before further downstream processing.

BACKGROUND OF THE INVENTION

Polypeptides or proteins may be made by recombinant DNA technology usingbacterial cells (e.g. Escherichia coli) as hosts. Thus, bacterial cellsmay be transformed with plasmid DNA encoding said polypeptide orprotein. The bacteria are thereby enabled to express quantities of thepolypeptide in either the cytoplasm, or the periplasm or theextracellular space. As the bacteria can be grown in large amounts usinglarge-scale fermentation processes, it is possible to produce largequantities of the polypeptide in this way.

Secretion of polypeptides or proteins into the periplasm has a number ofpotential advantages, including separation from cytoplasmic proteins,particularly proteases, avoidance of cytoplasmic toxicity, avoidance ofN-terminal methionine extension, and accumulation in a more oxidizingenvironment where disulfide-bond formation may proceed and the proteinmay fold into a soluble, biologically active confirmation.Superficially, it requires only that the desired protein be fused to asignal peptide (secretory leader) at its N-Terminus although, theefficiency of secretion is likely also influenced by structural featuresof the protein, as well as its usual location within the cell.

E. coli is well equipped to secrete proteins through the cytoplasmicmembrane into the periplasm, and this approach has been used widely todirect heterologous proteins out of the cell cytoplasm. It has beenparticularly successful in enabling the production of biologicallyactive antibody fragments and this approach now challenges theproduction of whole antibodies in animal cell culture.

Secretion of heterologous proteins is rarely 100% efficient and, inseveral cases, unprocessed precursor protein with the secretory leaderattached accumulates within the cell. This observation suggests that oneor more components of the secretory apparatus can limit the export ofthese proteins.

One notable feature of the literature on protein secretion in E. coli isthe frequency of reports in which the protein is recovered directly fromthe growth medium. This can be highly advantageous, but raisesunresolved questions about the underlying mechanisms. There are reportsshowing that proteins are often released into the medium by non specificleakage from, or lysis of, the cell, and not by specific translocationthrough the outer membrane. This phenomenon is not understood and ishighly protein-sequence specific, being extensive with some antibodyfragments and insignificant with other polypeptides.

Secretion of proteins into the periplasm is a useful route and can leadto the rapid isolation of a protein for biological evaluation. Itsapplication on industrial scale is currently limited by the generalunavailability of efficient, scaleable methods for selective release ofperiplasmic proteins from the cell.

If the recovery of a recombinant protein from the periplasm can beachieved without contamination by cytoplasmic proteins, subsequentpurification steps are simplified very much, since, for instance in E.coli, only 7 out of the 25 known cellular proteases and about 4-8% ofthe total cell protein are located in the periplasm (Swamy et al.,Baneyx et al.).

There are various frequently used methods for selective release ofperiplasmic proteins. One is cell permeabilization involving chemicalssuch as chloroform, guanidine-HCl, Cetyl-trimethyl-ammonium-bromide/CTABor detergents such as Triton X-100 and glycine. Others arepermealization using lysozyme/EDTA treatment or application of osmoticshock. These release methods are suitable also for large scalepreparation and have been used in many different modifications on a widerange of expression systems with varying degrees of success.

The state of the art methodology on periplasmic release of proteins isdocumented for example in the following literature:

-   Swamy et al., J. Bacteriol. 147, 1027-1033, 1982-   Baneyx et al., J. Bacteriol. 173, 2696-2703, 1991-   Blight et al., TibTech 12, 450-455, 1994-   Barbero et al., Journal of Biotechnology 4, 255-267, 1986-   Pierce et al., Journal of Biotechnology 58, 1-11, 1997-   French et al., Enzyme & Microbial Technology 19, 332-338, 1996-   Naglak et al., Enzyme & Microbial Technology 12, 603-611, 1990-   Nossal et al., J. Biol. Chem 241 (13), 3055-3062, 1966-   Neu et al., J. Biol. Chem 240, 3685-3692, 1965-   Hsiung et al., Bib/Technology 4, 991-995, 1986-   Carter et al., Bio/Technology 10, 163-167, 1992-   Georgiou et al., Biotechnol. Bioeng. 32, 741-748, 1988-   Aristidou et al., Biotechnolgy Letters 15 (4), 331-336, 1993-   Chaib et al., Biotechnology Techniques 9 (3), 179-184, 1195-   Ames et al., J. Bacteriol. 160, 1181-1183, 1984-   Gellerfors et al., J. Pharm. Biomed. Anal. 7, 2, 173-83, 1989-   Chapman et al, Nature Biotechnology 17, 780-783, 1999-   Voss et al., Biochem. J. 298, 719-725, 1994-   WO 01/94585-   U.S. Pat. No. 4,845,032-   U.S. Pat. No. 4,315,852

Whereas recombinant techniques can be employed to produce high yields ofa crude polypeptide, the isolation and purification of the polypeptiderequires sophisticated and extensive procedures.

In a typical isolation procedure, the fermentation harvest broth isadjusted to a neutral pH (e.g pH 6.5-7.5) by addition of acid orcaustic. Thereafter, the bacterial cells are removed e.g. bycentrifugation or microfiltration to leave a liquid supernatant,containing unwanted soluble by-products, which is discarded. Theresultant bacterial cell mass is resuspended in an appropriate medium,e.g. a suitable buffer, and the cells are disrupted to extract andisolate the product.

Laborious extraction and isolation procedures are usually carried out inorder to separate the polypeptide of interest from as much fermentationby-products and other contaminants as possible to ensure that subsequentpurification steps proceed in an as efficient manner as possible.

Purification steps known in the art generally comprise precipitation andchromatographic separation techniques, and sometimes require additionalsteps like diafiltration and/or concentration procedures, which arelaborious and may lead to lower yields of extracted polypeptide andhigher production costs.

The extraction, isolation and purification of a polypeptide implicateslosses of material or biological activity at every stage of the process.

Proteolysis, i.e. degradation of the polypeptides by proteolyticenzymes, usually occurring after disruption of the bacterial cells, butalso observed in vivo, is considered to be one of the main causes ofprotein loss. This adventitious proteolysis is a technical problem whichrequires modifications of the methodology to minimize the degradation ofthe polypeptide of interest.

One way of keeping the rate of proteolysis low, is generally to performthe harvest, extraction, isolation and purification procedures atreasonably low temperatures and as fast as possible. Accordingly,relevant text books and standard protocols on isolation and purificationof polypeptides in general teach to proceed without unnecessary delaysand interruptions (Protein Protocols, Ed. J. M. Walker, Humana PressInc., January 1998).

Accordingly, there is a need for novel processes that enable theextraction of recombinant polypeptides of interest from bacterial cellsin a high yielding and cost-effective manner.

The present invention complies with the above mentioned needs byproviding novel methods for the preparation of recombinant polypeptides.

In the context of the present invention it has surprisingly been foundthat the yield of a recombinant polypeptide expressed in a bacterialhost comprising a periplasm can be increased by interrupting theisolation process after fermentation and put on hold the furtherprocessing of the fermentation harvest broth before the subsequent stepsof extraction and isolation of said polypeptide are performed.

SUMMARY OF THE INVENTION

The present invention relates to a process for the preparation of arecombinant polypeptide, comprising the steps of

a) fermenting a prokaryotic host cell comprising a periplasm and beingtransformed with a recombinant expression system capable of effectingsecretion of the polypeptide into the periplasm, which fermentation isperformed in a fermentation medium under conditions such that thepolypeptide is secreted into the periplasm of the host cell, and

b) interrupting the further processing of the fermentation harvest brothand maintaining it under defined conditions of temperature and pH priorto extraction.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references cited hereinare hereby incorporated by reference in their entirety.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA are used. Thesetechniques are well known and are explained in, for example, CurrentProtocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M.Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes 1 and 11, 1985(D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription andTranslation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986(R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press);Perbal, 1984, A Practical Guide to Molecular Cloning; the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wuand Grossman, and Wu, eds., respectively).

The present invention relates to a process for the preparation of arecombinant polypeptide, comprising the steps of

a) fermenting a prokaryotic host cell comprising a periplasm and beingtransformed with a recombinant expression system capable of effectingsecretion of the polypeptide into the periplasm, which fermentation isperformed in a fermentation medium under conditions such that thepolypeptide is secreted into the periplasm of the host cell, and

b) interrupting the further processing of the fermentation harvest brothand maintaining it under defined conditions of temperature and pH priorto extraction.

The invention is based on the surprising finding that the yield of arecombinant polypeptide expressed in a bacterial host comprising aperiplasm can be increased by interrupting the isolation process afterfermentation and put on hold the further processing of the fermentationharvest broth before the subsequent steps of extraction and isolation ofsaid polypeptide are performed.

The expression of the polypeptide is performed in a suitable prokaryotichost cell comprising a periplasm and being transformed with arecombinant expression system capable of effecting secretion of thepolypeptide into the periplasm (described e.g. in Skerra and Plickthun,Science 240, 1038-1041, 1988).

The fermentation of the polypeptide is performed in a fermentationmedium under appropriate conditions such that the polypeptide issecreted into the periplasm of the host cell (described e.g. in Skerraand Plückthun, Science 240, 1038-1041, 1988).

As used herein, the term “fermentation” is understood to mean growing ahost cell in an appropriate fermentation medium for an appropriateperiod of time under appropriate conditions such that the polypeptide isproduced by the host cell and secreted into the periplasm.

Said interruption of the further processing may be accomplished, forexample, by maintaining, retaining, keeping or storing the fermentationharvest broth for at least one hour under appropriate conditions whichensure as far as possible the integrity of the produced polypeptide,i.e. that it is not degraded or otherwise impaired in function orstructure. This can be achieved, for instance, by maintaining,retaining, keeping or storing the fermentation harvest broth eitherdirectly in the fermentation tank (fermenter), or transferring saidfermentation harvest broth into another tank or any other suitablecontainer after collection from the fermenter. Furthermore, thefermentation harvest broth may be stirred periodically or continuouslyduring the interruption step.

Accordingly, in a preferred embodiment, step b) of the above process isperformed in the fermenter.

Usually, with regard to the desired increase of product yield, therewill be a relationship between the duration of the interruption step andthe temperature applied. A low temperature will require a longer timeperiod whereas at a higher temperature a shorter time period will besufficient in order to achieve similar results. The optimal parametersare dependent on the expressed polypeptide, the host cell and theproduction conditions.

Accordingly, in a preferred embodiment, a process for the preparation ofa recombinant polypeptide is provided, wherein the further processing ofthe fermentation harvest broth is interrupted for a period of at leastabout one hour, e.g. for a period of one hour.

Preferably, the further processing of the fermentation harvest broth isinterrupted for a period of about one hour to about 72 hours, e.g. for aperiod of one hour to 72 hours. Further processing may be interruptedfor any period within this range.

More preferably, the further processing of the fermentation harvestbroth is interrupted for a period of about 12 hours to about 48 hours,e.g. for a period of 12 hours to 48 hours. Further processing may beinterrupted for any period within this range.

Most preferably, the further processing of the fermentation harvestbroth is interrupted for a period of about 12 hours, about 24 hours orabout 48 hours., e.g. for a period of 12 hours, 24 hours or 48 hours.

In a further preferred embodiment, a process for the preparation of arecombinant polypeptide is provided, wherein the interruption of thefurther processing of the fermentation harvest broth is performed at atemperature of about 2° C. to about 65° C., e.g. at a temperature of 2°C. to 65° C. Further processing may be interrupted at any temperaturewithin this range.

More preferably, further processing of the fermentation harvest broth isinterrupted at a temperature of about 4° C. to about 25° C., e.g. at atemperature of 4° C. to 25° C. Further processing may be interrupted atany temperature within this range.

Most preferably, further processing of the fermentation harvest broth isinterrupted at a temperature of about 4° C., about 10° C., about 15° C.,about 20° C. or about 25° C., e.g. at a temperature of 4° C., 10° C.,1-5° C., 20° C. or 25° C.

In a further preferred embodiment, a process for the preparation of arecombinant polypeptide is provided, wherein further processing of thefermentation harvest broth is interrupted for a period of about 12hours, about 24 hours or about 48 hours at a temperature of about 4° C.,about 10° C., about 15° C., about 20° C. or about 25° C., e.g. for aperiod of 12 hours, 24 hours or 48 hours at a temperature of 4° C., 1.0°C., 15° C., 20° C. or 25° C.

In another preferred embodiment, a process for the preparation of arecombinant polypeptide is provided, wherein the pH value of thefermentation harvest broth is maintained between about 4 to about 10during step b), e.g. between 4 to 10. The fermentation harvest broth maybe maintained at any pH value within this range.

More preferably, the pH value of the fermentation harvest broth ismaintained between about 5 to about 9 during step b), e.g. between 5 to9. The fermentation harvest broth may be maintained at any pH valuewithin this range.

Even more preferably, the pH value of the fermentation harvest broth ismaintained between about 6 to about 8 during step b), e.g. between 6 to8. The fermentation harvest broth may be maintained at any pH valuewithin this range.

Most preferably, the pH value of the fermentation harvest broth ismaintained at about 7, e.g. at 7 during step b).

In practising the present invention it is not necessary to stick toexact values of temperature, pH and time, i.e. with respect to the aboveembodiments said values are to be understood as approximate or meanvalues. The present invention is workable within a broad range ofconditions and allows some variation. A skilled person will know thatsome variation around given values is possible and in practice sometimeseven unavoidable. Hence, during the interruption step period e.g. thepreset pH value may change slightly and may be readjusted as appropriateor necessary. Also the applied temperature may vary to some extent, e.g.when the interruption step is performed by incubating the fermentationharvest broth in a cold storage room. In such a cold storage room thepreset temperature normally varies within a certain tolerable range,e.g. from 2° C. to 8° C. with an average mean value of e.g. 4° C. or 5°C. over a certain time period. Therefore, within the context of thepresent invention, a given pH of e.g. 4 or a given temperature of e.g.25° C. does not mean an exact pH of 4.0 or an exact temperature of 25.0°C.

Before starting the interruption period, the appropriate conditions suchas, for instance, suitable temperature and pH of the fermentationharvest broth are adjusted according to requirements determined by theexpressed polypeptide. The most suitable conditions for a specificpolypeptide will be either known or can be easily determined applyingstandard methods known in the art (Protein Protocols, Ed. J. M. Walker,Humana Press Inc., January 1998).

After the interruption period the fermentation harvest broth usuallywill be extracted in order to isolate the recombinant polypeptide andseparate it from cellular material and unwanted fermentationby-products. Frequently used methods for selective release ofperiplasmic proteins are among others lysozyme and/or EDTA treatmentoptionally followed by osmotic shock treatment or any other suitablerelease procedure like pH or temperature incubation for a specified timeperiod.

In the context of the present invention it is to be understood that stepb) of the above process does not have to be necessarily performeddirectly following step a). Additional process steps may be performedbetween step a) and b). For instance, a partial extraction of thefermentation harvest broth prior to the application of the interruptionperiod is encompassed by the present invention. As long as not thecomplete fermentation harvest broth is further processed, theinterruption period will result in an increased polypeptide yield.

In addition, the fermentation harvest broth may be washed and/orconcentrated in order to reduce the volume of the material and simplifyits further processing, e.g. by centrifugation using a disk stackseparator, microfiltration, flocculation and sedimentation or byprecipitation and filtration, resulting in a wet cell paste. The extentof concentration may vary from partially reducing the volume for just asmall percentage to the maximum possible or appropriate, which may e.g.be determined by the desired final consistency in terms of percent ofwet weight. The concentration of the fermentation harvest broth may bedone before or after the interruption step, but preferably is donebefore.

Therefore, in a preferred embodiment, a process for the preparation of arecombinant polypeptide is provided, comprising the steps of

a) fermenting a prokaryotic host cell comprising a periplasm and beingtransformed with a recombinant expression system capable of effectingsecretion of the polypeptide into the periplasm, which fermentation isperformed in a fermentation medium under conditions such that thepolypeptide is secreted into the periplasm of the host cell, and

b) interrupting the further processing of the fermentation harvest brothand maintaining it under defined conditions of temperature and pH priorto extraction, and

wherein the fermentation harvest broth is concentrated prior to step b).

Preferably, the fermentation harvest broth is concentrated bycentrifugation or microfiltration prior to step b).

A number of prokaryotic host cells comprising a periplasm may be used topractice the present invention.

Accordingly, in a preferred embodiment, a process for the preparation ofa recombinant polypeptide is provided, comprising the steps of

a) fermenting a prokaryotic host cell comprising a periplasm and beingtransformed with a recombinant expression system capable of effectingsecretion of the polypeptide into the periplasm, which fermentation isperformed in a fermentation medium under conditions such that thepolypeptide is secreted into the periplasm of the host cell, and

b) interrupting the further processing of the fermentation harvest brothand maintaining it under defined conditions of temperature and pH priorto extraction, and

wherein the prokaryotic host cell is a Gram-negative bacterium.

More preferably, the Gram-negative bacterium is selected from the groupconsisting of Escherichia sp., Pseudomonas sp., Enterobacter sp.,Erwinia sp., Campylobacter sp., Proteus sp., Aeromonas sp. andVitreoscilla sp.

Most preferred is the use of Escherichia coli. Particularly suitable E.coli strains are E. coli K and B strains such as e.g. E. coli K12 or E.coli BL21.

The process of the present invention is broadly applicable and is notlimited to the preparation of particular recombinant polypeptides orproteins.

However, in a preferred embodiment, the present invention provides aprocess for the preparation of a recombinant polypeptide, comprising thesteps of

a) fermenting a prokaryotic host cell comprising a periplasm and beingtransformed with a recombinant expression system capable of effectingsecretion of the polypeptide into the periplasm, which fermentation isperformed in a fermentation medium under conditions such that thepolypeptide is secreted into the periplasm of the host cell, and

b) interrupting the further processing of the fermentation harvest brothand maintaining it under defined conditions of temperature and pH priorto extraction, and

wherein the recombinant polypeptide is an antibody, a hormone or animmunomodulating agent.

More preferably, the recombinant polypeptide is a growth hormone, agrowth factor, an interferon, a cytokine, an enzyme, an enzyme inhibitoror an antibody fragment.

Most preferably, the recombinant polypeptide is a Fab-fragment, humangrowth hormone, interferon alpha-2b or granulocyte colony-stimulatingfactor.

The invention is further described by reference to the followingexamples. These examples are provided for illustration purposes and arenot intended to be limiting.

EXAMPLES

In the Examples the following abbreviations are used:

aa=amino acid

A=peak area

AEX=anion exchange chromatography

BH=bed height

C=conductivity (mS/cm)

CAP.P=capture pool

CE=clarified crude extract

CP=pellet after centrifugation

CR=cell resuspension

CV=column volume

DBE=direct broth extraction

DR=diaretentate

DSP=down stream processing

EBA=expanded bed adsorption

EXT=extraction

Fab′=antibody Fab′ fragment

GAC=Glutaryl-7-ACA-Acylase

HB=harvest broth

HCP host cell proteins

HG=homogenate

HIC=hydrophobic interaction chromatography

IPC=In Process Controls

MBR=Master Batch Record

MF=microfiltration

N=theoretical plates

p=pressure (bar)

P=protein

PCM=packed cell mass

PEI=polyethyleneimine

PS=primary separation

(a)RPC=(acid) reversed phase chromatography

rpm=rounds per minute

Rs=resolution

SDS-PAGE=sodium dodecylsulfate polyacrylamide gel electrophoresis

SEC=size exclusion chromatography

ss=stainless steel

T=temperature (° C.)

t=time (h)

TMP=transmembrane pressure

UF/DF=ultrafiltration/diafiltration

V=volume (L)

W=weight (kg)

wBM=wet biomass (=BFM)

WBR=purified water

WFI=water for injection

Example 1 Periplasmic Expression of an Antibody Fab′-Fragment (Fab′)

The light and heavy chain of a humanized antibody Fab′-fragment havingspecificity for human tumor necrosis factor-alpha (disclosed in WO01/94585) is cloned and expressed in E. coli K12. A vector is usedenabling the sequential expression of light and heavy chain each asN-terminal fusions with a signal sequence under common control of asuitable promoter.

After fermentation of the humanized Fab′ producing E. coli K12 strainthe harvest broth is clarified by centrifugation (Wesffalia CSC6 diskseparator, 100 Uh, 15000×g), then the isolated cell paste resuspendedimmediately or after a specified interruption period (time, temperature,wet weight consistency) with extraction buffer (200 mM TRIS/HCl pH 7.4,20 mM EDTA) to the original harvest volume (final concentration 100 mMTRIS/HCl, 10 mM EDTA) and each preparation extracted 30 min at 25° C.After the 30 min extraction an analytical aliquot of each cellsuspension preparation is clarified immediately by centrifugation(Beckman Avanti J25 I, Rotor 25.5, 20.000 g) plus 0.2 pjm filtration(PALL Acrodisc 32 syringe 0.2 μm filter). All clarified extracts areanalysed for Fab′ (aRPC) and total protein content (Bradford).

The results in table 1 show that the Fab′ extraction yield can beincreased by including an interruption step after clarification andprior to extraction. TABLE 1 Relative Interruption step extraction yieldSample h ° C. % 1 0 — 100 2 24  4 168 3 48  4 203 4 24 25 451 5 48 25621

Example 2 Periplasmic Expression of Recombinant Human Growth Hormone(rhGH)

The structural gene of human growth hormone is cloned and expressed inE. coli K12 using a vector enabling the expression of rhGH as anN-terminal fusion protein with a signal sequence under control of asuitable promoter. The signal sequence is cleaved during the export intothe periplasmic space of the host cell leaving a native polypeptidesequence in the periplasm.

After fed batch fermentation the rhGH containing E. coli harvest brothis adjusted to pH 5 with sulfuric acid and immediately cooled down to5-15° C. The low pH as well as the low temperature help to inactivateendogenous proteases and aminopeptidases. Then the treated harvest brothis concentrated to about half of the original volume by subsequentcentrifugation (Wesffalia separator CSC6, 200 L/h, 15000×g) ormicrofiltration (MF, 0.2 μm Hydrosart/Sartorius, TMP about 1 bar) andsubsequently washed with process water till a final conductivity of 3-5mS/cm.

Without or after a specified interruption step (24 hours or 48 hours at4° C. or 20° C.) the concentrated cell paste and the harvest brothitself (DBE without prior concentration/washing step) are conditionedwith sucrose/EDTA stock solution to reach a final concentration of 1 mMEDTA, 200 g/L sucrose and about 10-20% wet cell weight. After one hourincubation by gentle stirring at 2-8° C. the conditioned cell suspensionis diluted into cold water (osmotic shock by 1+4 dilution) and theincubation continued for an additional hour. After this osmotic shockpolyethyleneimine (Polyethyleneimine 50%, BASF) is added to a finalconcentration of 0.05% and the pH adjusted to 7.5 with sulfuric acid.Subsequent centrifugation (Wesffalia separator CSC6) and filtration (0.3μm Polygard plus 0.2 μm Durapore/Millipore) gives a clear proteinsolution with 0.1-0.3 mg rhGH/mL with a purity of >20% in respect tototal Bradford protein.

The rhGH containing extract is then adjusted to a final conductivity of3-5 mS/cm (or by dilution or by diafiltration over a 5-10 kD Biomaxmembrane, Millipore) and captured by anion exchange chromatography(Q-HyperD ° F., Biosepra, load capacity >50 mg total protein/ml packedresin, linear flow 3-5 cm/min, bed height 10-20 cm).

The rhGH extraction yield can be increased by including an interruptionstep after fermentation/clarification and prior to extraction.

Example 3 Periplasmic Expression of Recombinant Human InterferonAlpha-2B (rhIFN α-2B)

rhIFN α-2B is produced by fermentation of the recombinant E. coli K12W3110 strain under the control of the Pgac promoter. The target proteinis expressed as N-terminal fusion with the signal peptide of thebacterial Glutaryl-ACA-Acylase (GAC) from Pseudomonas diminuta CCM 3987(described in CS 278515, CS 260068).

As in example 2 the harvest broth is initially washed with process waterat constant volume and then concentrated by subsequent centrifugation(Wesffalia separator CSC6, 200 L/h, 15000×g) or microfiltration (MF, 0.2μm Hydrosart/Sartorius, TMP about 1 bar) until a final conductivity ofabout 4 (range 3-5) mS/cm and a final consistency of 45±5% wet weight.Further processing is performed with or without a subsequentinterruption step, i.e. with or without maintaining the concentratedcell paste or the harvest broth itself (DBE without priorconcentration/washing step) for 24 or 48 hours at a temperature of 4° C.or 20° C. Afterwards, an EDTA/sucrose stock solution is added to thewashed cell paste or harvest broth to reach a final concentration of 10mM EDTA, 200 g/L sucrose and 10-20% wet cell weight, and the slurry isadjusted to pH 8.0 with NaOH. After one hour incubation by gentlestirring at 2-8° C. the conditioned cell suspension is diluted into coldwater (osmotic shock by 1+4 dilution) and the incubation continued foran additional hour, then clarified similar as in the rhGH experiment(example 2) via Polyethylene-imine flocculation and subsequentcentrifugation and filtration resulting in a clear protein solution withapproximately 0.005-0.025 mg rhIFN α-2B per mL extract (estimated bydensitometric SDS-PAGE plus Western detection). For product capture theclear extract is adjusted to 3.5-4.5 mS/cm (or by dilution or bydiafiltration over a 5-10 kD Biomax membrane, Millipore), adjusted to pH5 with diluted CH₃COOH and loaded on an equilibrated S Ceramic HyperD Fcolumn (S Ceramic HyperD F, Biosepra, load capacity 30-70 mg protein/mlresin, 2-6 cm/min, EQ-buffer 20 mM Na-acetate+70 mM NaCL pH 5.0). Aftera washing step with equilibration buffer the rhIFN α-2B is eluted withapproximately 2 CV elution buffer (20 mM Na-acetate+175 mM NaCL pH 5.0).In the capture pool the target protein is quantified by aRPC and thepurity estimated by product content versus total protein measured withthe Bradford method.

The rhIFN α-2B extraction yield can be increased by including aninterruption step after fermentation/clarification and prior toextraction.

1. A process for the preparation of a recombinant polypeptide,comprising the steps of a) fermenting a prokaryotic host cell comprisinga periplasm and being transformed with a recombinant expression systemcapable of effecting secretion of the polypeptide into the periplasm,wherein the fermentation is performed in a fermentation medium underconditions such that the polypeptide is secreted into the periplasm ofthe host cell, and b) interrupting the further processing of thefermentation harvest broth and maintaining it under defined conditionsof temperature and pH prior to extraction.
 2. A process according toclaim 1, wherein the further processing of the fermentation harvestbroth is interrupted for a period of at least about one hour.
 3. Aprocess according to claim 1, wherein the further processing of thefermentation harvest broth is interrupted for a period of about one hourto about 72 hours.
 4. A process according to claim 1, wherein thefurther processing of the fermentation harvest broth is interrupted fora period of about 12 hours to about 48 hours.
 5. A process according toclaim 1, wherein the further processing of the fermentation harvestbroth is interrupted for a period of about 12 hours, about 24 hours orabout 48 hours.
 6. A process according to claim 1, wherein theinterruption of the further processing of the fermentation harvest brothis performed at a temperature of about 2° C. to about 65° C.
 7. Aprocess according to claim 1, wherein the interruption of the furtherprocessing of the fermentation harvest broth is performed at atemperature of about 4° C. to about 25° C.
 8. A process according toclaim 1 wherein the interruption of the further processing of thefermentation harvest broth is performed at a temperature of about 4° C.,about 10° C., about 15° C., about 20° C. or about 25° C.
 9. A processaccording to claim 1, wherein the pH value of the fermentation harvestbroth is maintained between about 4 to about 10 during step b).
 10. Aprocess according to claim 1, wherein the pH value of the fermentationharvest broth is maintained between about 5 to about 9 during step b).11. A process according to claim 1 wherein the pH value of thefermentation harvest broth is maintained between about 6 to about 8during step b).
 12. A process according to claim 1, wherein the pH valueof the fermentation harvest broth is maintained at about 7 during stepb).
 13. A process according to claim 1, wherein the further processingof the fermentation harvest broth is interrupted for a period of about12 hours, about 24 hours or about 48 hours at a temperature of about 4°C., about 10° C., about 15° C., about 20° C. or about 25° C.
 14. Aprocess according to claim 1, wherein the fermentation harvest broth isconcentrated prior to step b).
 15. A process according to claim 1,wherein the fermentation harvest broth is concentrated by centrifugationor microfiltration prior to step b).
 16. A process according to claim 1,wherein step b) is performed in the fermenter.
 17. A process accordingto claim 1, wherein the prokaryotic host cell is a Gram-negativebacterium.
 18. A process according to claim 17, wherein theGram-negative bacterium is selected from the group consisting ofEscherichia sp., Pseudomonas sp., Enterobacter sp., Erwinia sp.,Campylobacter sp, Proteus sp., Aeromonas sp. and Vitreoscilla sp.
 19. Aprocess according to claim 17, wherein the Gram-negative bacterium isEscherichia coli.
 20. A process according to claim 1, wherein therecombinant polypeptide is an antibody, a hormone or an immunomodulatingagent.
 21. A process according to claim 1, wherein the recombinantpolypeptide is a growth hormone, a growth factor, an interferon, acytokine, an enzyme, an enzyme inhibitor or an antibody fragment.
 22. Aprocess according to claim 1 wherein the recombinant polypeptide is aFab-fragment, human growth hormone, interferon alpha-2b or granulocytecolony-stimulating factor.
 23. (canceled)